Haemophilus ducreyi is a strict human pathogen that causes chancroid, a genital ulcer disease (GUD) that facilitates the transmission of the human immunodeficiency virus (HIV-1). To study the biology of H. ducreyi, we developed a human challenge model that closely simulates natural infection. In experimental and natural infection, H. ducreyi is found in the hostile environment of an abscess and resists phagocytosis. In the last funding period, we found that several hundred bacterial genes were differentially regulated in vivo relative to in vitro growth, suggesting that H. ducreyi senses and responds to the adverse host environment. H. ducreyi contains only two systems known to respond to extracytoplasmic stress: the 2-component regulator CpxRA and the alternative sigma factor, RpoE. Several H. ducreyi homologues of CpxRA-specific effectors were upregulated, while many homologues of RpoE-specific effectors were downregulated in vivo, suggesting that that CpxRA and RpoE systems are linked in H. ducreyi and function in a coordinated fashion to respond to environmental stresses mounted by the host. We found that CpxRA controlled the expression of several major virulence determinants of H. ducreyi and that an intact CpxRA system is absolutely required for virulence in human volunteers. We hypothesize that H. ducreyi senses the host environment via CpxRA and RpoE, that both CpxRA and RpoE function during infection to combat stresses encountered in vivo and control the production of virulence determinants, that constitutively active expressers or deletion mutants in the CpxRA or RpoE systems will be attenuated for virulence and that known or novel virulence determinants will be differentially regulated by CpxRA and RpoE during infection. To test these hypotheses, our specific aims include: 1) evaluation of cpxR, rpoE, rseA and rseC deletion mutants and strains that express constitutively activated CpxR and RpoE for virulence in human volunteers; 2) determination of the mechanism(s) underlying the contributions of the CpxRA and RpoE systems to pathogenesis in models relevant to human infection; 3) identification of novel virulence determinants controlled by the CpxRA and RpoE pathways and evaluation of their role in infection in the in vivo (Aim1) and in vitro (Aim 2) models. Our proposal offers the unique opportunity to study the contributions of two interrelated stress response systems to the survival of a pathogen in humans and will lead to the identification of novel virulence determinants. (Note that due to NIAID policy, the human challenge trials will be supported by other funds, not this R01.) PUBLIC HEALTH RELEVANCE: Haemophilus ducreyi causes a genital ulcer disease called chancroid, which facilitates HIV transmission. H. ducreyi turns on the expression of many of its genes when it infects the human host. Our study is designed to examine how the bacterium uses stress response systems to adapt to the human host. Chancroid can be treated with only three types of antibiotics. The genes controlled by the stress response systems may prove to be targets of vaccines or alternative treatments for H. ducreyi infection.